Lectins and Immunomodulation
The cytotoxic cells of the immune system, cytolytic T cells (CTL), natural killer (NK) cells and macrophages, can seek out and ultimately lyse tumor cells either spontaneously or more often after appropriate activation. Spontaneous cytotoxic activity against tumor cells is mainly a result of NK cells. Various cytokines, alone or in combination, have been shown to augment anti-tumor activity : IL-2, IL-7, IL-12 and IFN-γ induce cytotoxic activity in NK and T-cells while IFN-γ and TNFα are potent activators of macrophages and monocytes. Most of the studies that have demonstrated these effects have been confined to in vitro systems although recently the anti-tumor effect of some of these cytokines has also been demonstrated in vivo in animals and also in humans. Lymphocytes cultured in the presence of high amounts of IL-2 are refered to as lymfokine-activated killer (LAK) cells. LAK cells are characterised by their ability to kill NK-resistant tumor cells without major histocompatibility complex (MHC) restriction. Although both NK and T cells are responsible for LAK activity, the former are responsible for mediating most of the activity. Macrophages and monocytes are known to accumulate around tumors. Following the TNFα and IFN-γ stimulated activation of these cells it is predicted that a local release of cytokines would occur from these activated cells directly into the tumor. This in turn would be expected to induce apoptosis and ultimately cause death of the tumor cells.
Besides cytokines, a variety of natural or synthetically produced protein mixtures have been reported to exert immunomodulating properties. The commercially available mistletoe extracts belong to this category of agents. Biochemical analysis has shown that the immunomodulating capacity is due to the presence of mistletoe lectins (ML-I, ML-II and ML-III) in the extracts.
Mistletoe Extracts and Use in Cancer Therapy.
Mistletoe extracts have been used in cancer therapy for more than 80 years, particularly in clinics in Austria, Switzerland and Germany. Use of these extracts has been heavily criticized by practitioners of traditional “school medicine” due to the lack of knowledge concerning the actual nature of active anti-cancer components in mistletoe. Recent work has now clearly shown that purified lectins (ML-I, ML-II, ML-III) present in mistletoe extracts possess both immunomodulatory and cytotoxic properties. The arguments which have been raised by advocates of “school medicine” are thus to a large extent no longer valid.
Treatment methods employing known mistletoe extracts have several disadvantages, however. In the first place, commercially available extracts vary greatly in terms of their composition, thus making both study and treatment regimes unreliable. Furthermore, it is generally believed that mistletoe is toxic. The most common method of clinically treating cancer patients with mistletoe extracts, therefore, has been through subcutaneous injection. Recent work, however, has shown that serum glycoproteins effectively bind to and thus will minimise the effects of mistletoe lectins injected into cancer patients. This suggests that the subcutaneous route is probably not very effective. An alternate delivery method is therefore desirable.
Dietary Lectins and Tumor Growth
As has been shown in a series of publications since 1994 (see references), the growth of intraperitoneal or subcutaneous non-Hodgkin lymphoma tumors in NMRI mice can be reduced by feeding the animals a diet containing the lectin present in raw kidney bean (Phaseolus vulgaris), phytohaemagglutinin (PHA) or mistletoe lectin (ML-I). Other observations have shown that the development of a subcutaneous plasmacytoma tumor (MPC-11) in Balb/c mice can be greatly reduced by feeding a diet that included PHA. The lectins, being resistant to the proteolytic effect of gut enzymes, retain their biological activity in the alimentary canal. When added to the diet of experimental animals they bind to the surface of the gut mucosa and induce a fully reversible, dose-dependent hyperplastic growth of the small intestine. Simultaneously with the stimulated gut growth PHA induces an extensive absorption of amino acids and other nutrients from the intestinal lumen. Prior to the onset of hyperplastic growth, an extensive accumulation of extracellular polyamines occurs in the intestinal mucosa. Polyamines are molecules which play major roles in a series of important mechanisms controlling cell proliferation and, importantly, are involved in tumor growth.
In experiments to study the importance of the timing of feeding mice the PHA-containing diet with respect to when the tumor cells were injected, diets were changed on specific days. It was shown that the number of tumor cells was significantly lower when the lactalbumin-based (La) diet was replaced by one supplemented with PHA on the same day as tumor cells were injected. Recent experiments have shown that when PHA was added to the diet of mice bearing established NHL tumors then further growth was greatly retarded. Preliminary data have shown that PHA fed to rats causes a rapid increase in TNFα production. Accelerated cellular turnover within the transplanted NHL tumor as a response to oral intake of ML-1 was seen as increased numbers of apoptotic cells with an increased area of serpiginous irregular dead cells, and the non-viable cells occupied a two fold increased area in the mice fed the lectin. Apoptoses were more numerous in the tumors of mice fed ML-I and these were identified by nick end-labelling around areas of non-viable tumor cells, at the advancing edge of the tumor and within intense lymphoid aggregates. Morphological studies of tumor sections showed a greatly reduced incidence of tumor vascularisation indicating that ML-1 induces the production of anti-angiogenic factors. Recent experiments performed with purified ML-III have shown that feeding of the lectin to mice bearing established NHL tumors was extremely effective in reducing further tumor growth.
While it has thus been shown in animal models that the purified mistletoe lectins are well tolerated when administered orally, the purified mistletoe lectins are extremely expensive and time consuming to produce, making them less than ideal candidates as a treatment method. There is a need, therefore, for a preparation of mistletoe lectins and related treatment method that can be administered orally and that is inexpensive to produce.
Although it is generally believed that mistletoe is toxic, a recent study concerning the outcome of 1754 exposures has shown that accidental ingestion of the plant is not associated with profound toxicity. There are, however, components present in mistletoe extracts that do induce nausea. These nausea-inducing compounds, including alkaloids and viscotoxins, have been mistakenly regarded as essential to the medicinal effectiveness of mistletoe extracts. (See, for example, U.S. Pat. Nos. 5,637,563 and 5,547,674). As a result, no effort has been previously undertaken to produce a mistletoe preparation that can be satisfactorily administered orally to humans.
A procedure for producing purified mistletoe lectins is described by R. Eifler, K. Pfüller, W. Göckeritz and U. Pfüller in “Lectins : Biology, Biochemistry and Clinical Biochemistry” vol. 9 (1993) pp 141-151, which is incorporated by reference herein.
The applicant has recognized that the complex procedure used by Eifler et al to isolate and purify the three individual mistletoe lectins (ML-I, II and III) is not suitable for producing a therapeutic, orally ingestible lectin preparation, however. The rationale being:                (i) The applicant has shown that ML-1, when added to the diets of mice on the same day as tumor cell injection, reduces the mass of non-Hodgkin lymphoma tumors related to controls. A clear dose-response was observed. At the highest amount of ML-1 ingested a total ablation of tumors was seen in 25% of animals. Furthermore, the applicant has recently shown that when purified ML-III was fed in the diet to mice 5 days after subcutaneous injection of Krebs II cells (non-Hodgkin lymphoma), then the growth of the established tumor was arrested. Since the three lectins have different biological specificities (e.g. sugar binding) then they may well act better in concert than if supplied separately. Purifying the individual lectins is therefore counterproductive from a medicinal point of view.        (ii) the complete purification procedure for the three lectins is considerably time consuming, and        (iii) the lectin preparation according to the invention showed surprisingly, and contrary to the accepted belief in the art, that it could be taken orally. Thus a small contamination by other proteins would not represent any major problem since proteins other than the lectins would be subject to breakdown by digestive enzymes.        
The applicant has therefore modified and improved upon the procedure of Eifler et al. in order to arrive at a mistletoe lectin preparation that contains the lectins ML-I, ML-II and ML-III, possibly together with insignificant amounts of impurities, but which specifically excludes the nausea-inducing compounds otherwise present.